Nano-Regulation of Gene Expression in Chlamydomonas reinhardtii: Harnessing AuNPs for Remotely Switchable Lipid Biosynthesis via Antisense Oligonucleotides.

Antisense oligonucleotide (ASO)-mediated gene silencing has broad applications, spanning from biomedicine to agriculture, involving molecular biology, synthetic biology, and genetic manipulation. This research harnessed nanotechnology to augment ASO-mediated gene silencing, introducing a remotely switchable gene expression system for precise temporal control. We targeted lipid biosynthesis and accumulation enhancement in the photosynthetic eukaryote Chlamydomonas reinhardtii. Gold nanoparticles (AuNPs) transported double-stranded DNA (dsDNA), forming dsDNA-AuNP complexes. These complexes comprised 3'-thiolated sense strands attached to AuNPs and fluorescent antisense oligonucleotides. To avoid harmful laser effects on cells, we adopted a light-emitting diode (LED). Confocal microscopy confirmed dsDNA-AuNP internalization in C. reinhardtii. LED-triggered antisense release led to an 83% decrease in Citrate Synthase 2 (CIS 2) expression. Thiolated sense strand attachment postillumination inhibited antisense reannealing, enhancing gene silencing. This led to significant lipid body accumulation in cells, verified through fluorometric and fluorescence microscopy. This union of nanotechnology and ASO-mediated silencing provides gene regulation opportunities across sectors like biomedicine and agriculture. The system's remote switching capability underscores its potential in synthetic biology and genetic engineering. Our findings substantiate the utility of this approach for enhancing lipid biosynthesis in C. reinhardtii but also underscores its broader applicability to other organisms, fostering the development of novel solutions for pressing global challenges in energy, agriculture, and healthcare.

CAR-T Cell Therapy: From the Shop to Cancer Therapy.

Cancer is a worldwide health problem. Nevertheless, new technologies in the immunotherapy field have emerged. Chimeric antigen receptor (CAR) technology is a novel biological form to treat cancer; CAR-T cell genetic engineering has positively revolutionized cancer immunotherapy. In this paper, we review the latest developments in CAR-T in cancer treatment. We present the structure of the different generations and variants of CAR-T cells including TRUCK (T cells redirected for universal cytokine killing. We explain the approaches of the CAR-T cells manufactured ex vivo and in vivo. Moreover, we describe the limitations and areas of opportunity for this immunotherapy and the current challenges of treating hematological and solid cancer using CAR-T technology as well as its constraints and engineering approaches. We summarize other immune cells that have been using CAR technology, such as natural killer (NK), macrophages (M), and dendritic cells (DC). We conclude that CAR-T cells have the potential to treat not only cancer but other chronic diseases.

Curcumin Sensitizes 4T1 Murine Breast Cancer Cells to Cisplatin Through PAR4 Secretion.

BACKGROUND/AIM: Prostate apoptosis response 4 (PAR4), a tumour-suppressor protein, selectively induces apoptosis of cancer cells without affecting normal cells. Its soluble form is induced by secretagogues (e.g., chloroquine), and it induces apoptosis by interacting with the receptor of glucose-regulated protein 78, which is overexpressed in cancer cells. In this study, curcumin was analyzed as an inducer of PAR4 expression in 4T1 murine breast cancer cell. and its ability to induce PAR4 secretion in Balb/c mice. In addition, the cisplatin sensitizing effect of soluble PAR4 was analyzed. MATERIAL AND METHODS: The 4T1 cell line was treated in vitro using different concentrations of curcumin; cell viability was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and PAR4 expression by western blotting. The expression of soluble PAR4 in the serum of mice treated with intraperitoneal curcumin was analyzed using the dot-blot method. Moreover, MTT assay was used to analyze the effects of serum from curcumin-treated mice on cell viability. Tumor size was analyzed in mice treated with curcumin alone and in combination with cisplatin. RESULTS: Curcumin showed a dose- and time-dependent effects on cell viability on 4T1 cells, as well as increasing PAR4 expression. Compared with the control group (phosphate-buffered saline), mice treated with curcumin showed an increase in plasma PAR4. In the Balb/C tumor model, mice treated with curcumin and cisplatin showed greater tumor shrinkage than the control group. CONCLUSION: These results indicate that curcumin induces expression of soluble PAR4 and sensitizes tumor cells to cisplatin.

The Inflammatory Process Modulates the Expression and Localization of WT1 in Podocytes Leading to Kidney Damage.

BACKGROUND/AIM: Wilms' tumor 1 (WT1) is involved in the development of the urogenital system and is expressed in podocytes throughout life. Inflammation of renal glomeruli causes renal damage-induced nephrotic syndrome and steroid-resistant nephrotic syndrome have mutations in the WT1 gene. The aim of this work was to determine if the inflammatory process modulates the expression and localization of WT1 in podocytes that cause kidney damage using lipopolysaccharide (LPS)-treated mice as a sepsis model. MATERIALS AND METHODS: In investigation of renal damage, proteinuria and histology were analyzed. WT1 modulation was analyzed by indirect immunofluorescence, immunohistochemistry and western blot assays, and proinflammatory cytokines were analyzed by quantitative polymerase chain reaction assay. RESULTS: WT1 expression decreased most at 24 and 36 h after the induction of inflammation and phosphorylated WT1 was mainly localized in the cytoplasm, reduced nephrin mRNA expression and increased mRNA expression of tumor necrosis factor α and interleukin 1ß. CONCLUSION: These results indicate that the immune system plays an important role in the modulation of WT1, leading to kidney damage.

Inhibitory Effect of Cuphea aequipetala Extracts on Murine B16F10 Melanoma In Vitro and In Vivo.

Cuphea aequipetala (C. aequipetala) has been used in Mexican traditional medicine since prehispanic times to treat tumors. In this paper, we evaluated the antiproliferative and apoptotic effect of the methanolic and aqueous extracts of C. aequipetala on several cancer cell lines including the B16F10 cell line of murine melanoma and carried a murine model assay. In vitro assay analyzed the effect in the cellular cycle and several indicators of apoptosis, such as the caspase-3 activity, DNA fragmentation, phosphatidylserine exposure (Annexin-V), and induction of cell membrane permeabilization (propidium iodide) in the B16F10 cells. In vivo, groups of C57BL/6 female mice were subcutaneously injected with 5x105 B16F10 cells and treated with 25 mg/mL of C. aequipetala extracts via oral. Aqueous and methanolic extracts showed a cytotoxic effect in MCF-7, HepG2, and B16F10 cell lines. The methanolic extract showed more antiproliferative effect with less concentration, and for this reason, the in vitro experiments were only continued with it. This extract was able to induce accumulation of cells on G1 phase of the cell cycle; moreover, it was able to induce DNA fragmentation and increase the activity of caspase-3 in B16F10 cells. On the other hand, in the murine model of melanoma, the aqueous extract showed a greater reduction of tumor size in comparison with the methanolic extract, showing an 80% reduction versus one of around 31%, both compared with the untreated control, indicating a better antitumor effect of C. aequipetala aqueous extract via oral administration. In conclusion, the in vitro data showed that both C. aequipetala extracts were able to induce cytotoxicity through the apoptosis pathway in B16F10 cells, and in vivo, the oral administration of aqueous extract reduces the melanoma tumoral mass, suggesting an important antitumoral effect and the perspective to search for effector molecules involved in it.

shRNA-WT1 Potentiates Anticancer Effects of Gemcitabine and Cisplatin Against B16F10 Lung Metastases In Vitro and In Vivo.

BACKGROUND/AIM: High expression level of Wilm's tumor gene (WT1) in several types of tumors appears to confer disruption of apoptosis and resistance to chemotherapeutic drugs, and correlate with poor outcome. The aim of this work was to determine if down-regulation of WT1 expression results in decreased cell proliferation and the increased action of different types of drugs, both in vitro in B16F10 cells, and in vivo in C57BL/6 mice. MATERIALS AND METHODS: Inhibition of cell proliferation by short hairpin RNA against WT1 (shRNA-WT1), cisplatin, and gemcitabine in B16F10 cells in vitro was determined by the MTT assay and analysis of clonogenic survival. The apoptosis rate was determined by flow cytometry for annexin-V- fluorescein isothiocyante and propidium iodide. RESULTS: Compared to treatment with shRNA-WT1 alone, treatment with shRNA-WT1 in combination with drugs had a synergistic inhibitory effect on B16F10 cell proliferation, particularly for the combination of cisplatin and gemcitabine at their 25% cytotoxic concentrations in vitro. Furthermore, mice treated with shRNA-WT1 in combination with cisplatin and gemcitabine were protected in the same way as those treated with the drugs alone, but were in better physical condition. CONCLUSION: Decreased WT1 expression induces cell death and potentiates the action of anticancer drugs by inducing synergistic effects both in vitro and in vivo, which may be an attractive strategy in lung cancer therapy.

Molecular diagnosis of microbial copathogens with influenza A(H1N1)pdm09 in Oaxaca, Mexico.

BACKGROUND: Multiple factors have been associated with the severity of infection by influenza A(H1N1)pdm09. These include H1N1 cases with proven coinfections showing clinical association with bacterial contagions. PURPOSE: The objective was to identify H1N1 and copathogens in the Oaxaca (Mexico) population. A cross-sectional survey was conducted from 2009 to 2012. A total of 88 study patients with confirmed H1N1 by quantitative RT-PCR were recruited. METHODS: Total nucleic acid from clinical samples of study patients was analyzed using a TessArray RPM-Flu microarray assay to identify other respiratory pathogens. RESULTS: High prevalence of copathogens (77.3%; 68 patients harbored one to three pathogens), predominantly from Streptococcus, Haemophilus, Neisseria, and Pseudomonas, were detected. Three patients (3.4%) had four or five respiratory copathogens, whereas others (19.3%) had no copathogens. Copathogenic occurrence with Staphylococcus aureus was 5.7%, Coxsackie virus 2.3%, Moraxella catarrhalis 1.1%, Klebsiella pneumoniae 1.1%, and parainfluenza virus 3 1.1%. The number of patients with copathogens was four times higher to those with H1N1 alone (80.68% and 19.32%, respectively). Four individuals (4.5%; two males, one female, and one infant) who died due to H1N1 were observed to have harbored such copathogens as Streptococcus, Staphylococcus, Haemophilus, and Neisseria. CONCLUSION: In summary, copathogens were found in a significant number (>50%) of cases of influenza in Oaxaca. Timely detection of coinfections producing increased acuity or severity of disease and treatment of affected patients is urgently needed.

Silencing of Foxp3 delays the growth of murine melanomas and modifies the tumor immunosuppressive environment.

Forkhead box p3 (Foxp3) expression was believed to be specific for T-regulatory cells but has recently been described in non-hematopoietic cells from different tissue origins and in tumor cells from both epithelial and non-epithelial tissues. The aim of this study was to elucidate the role of Foxp3 in murine melanoma. The B16F10 cell line Foxp3 silenced with small interference Foxp3 plasmid transfection was established and named B16F10.1. These cells had lower levels of Foxp3 mRNA (quantitative real-time reverse transcription-polymerase chain reaction [0.235-fold]), protein (flow cytometry [0.02%]), CD25(+) expression (0.06%), cellular proliferation (trypan blue staining), and interleukin (IL)-2 production (enzyme-linked immunosorbent assay [72.35 pg/mL]) than those in B16F10 wild-type (WT) cells (P<0.05). Subcutaneous inoculation of the B16F10.1 cell line into C57BL/6 mice delayed the time of visible tumor appearance, increased the time of survival, and affected the weight of tumors, and also decreased the production of IL-10, IL-2, and transforming growth factor beta compared with mice inoculated with the B16F10 WT cell line. The B16F10.1 cells derived from tumors and free of T-cells (isolated by Dynabeads and plastic attachment) expressed relatively lower levels of Foxp3 and CD25(+) than B16F10 WT cells (P<0.05) in a time-dependent manner. The population of tumor-infiltrating lymphocytes of T CD4(+) cells (CD4(+), CD4(+)CD25(+), and CD4(+)CD25(+)Foxp3(+)) increased in a time-dependent manner (P<0.05) in tumors derived from B16F10 WT cells and decreased in tumors derived from B16F10.1 cells. Similar data were obtained from spleen cells. These results suggest that, in melanomas, Foxp3 partly induces tumor growth by modifying the immune system at the local and peripheral level, shifting the environment toward an immunosuppressive profile. Therapies incorporating this transcription factor could be strategies for cancer treatment.

WT1 shRNA delivery using transferrin-conjugated PEG liposomes in an in vivo model of melanoma.

The global incidence of melanoma is increasing. Mortality from melanoma is influenced primarily by metastasis in advanced stages of the disease. Current treatments are largely ineffective; thus, novel gene delivery approaches that target tumor-specific markers may be useful for the treatment of melanoma. Systemic administration of encapsulated RNA-interference plasmids targeted against tumor cells is a potential alternative therapy for cancer. Formulations of transferrin (Tf)-conjugated polyethylene glycol (PEG) liposomes loaded with short hairpin RNA (shRNA) against WT1 (Lip + RNAi + Tf), PEG liposomes loaded with shRNA against WT1 (Lip + RNAi), Tf-conjugated PEG liposomes loaded with pEGFP-N3 (Lip + GFP + Tf) and saline solution as negative control (untreated) were administered systemically to C57BL/6 mice implanted subcutaneously with a melanoma cell line. Tumor volume, body weight, tumor weight, survival and relative expression of WT1 were evaluated. No significant differences in net body weight were identified between groups. The tumor volume decreased from 7,871 mm3 (SD±2,087) in the untreated group to 5,981 mm3 (SD±2,099) in the Lip + RNAi + Tf group. The tumor weight was reduced, from 8.8 g (SD±0.30) in the untreated group to 5.5 g (SD±0.87) in the Lip + RNAi + Tf group. An increase of 37% in survival was also observed in the group treated with Lip + RNAi + Tf in comparison to the untreated group. Tumors treated with Lip + RNAi + Tf also showed a decrease in the mean relative expression of WT1 of 0.21 (SD±0.28) folds compared with 1.8 (SD±2.49) folds in untreated group, 1.34 (SD±0.43) folds in Lip + RNAi group and of 1.89 (SD±0.69) folds in Lip + GFP + Tf group. Systemic administration of transferrin-conjugated PEG liposomes loaded with shRNA against WT1 reduced WT1 expression and tumor size and increased survival.

CD133 antisense suppresses cancer cell growth and increases sensitivity to cisplatin in vitro.

The increased incidence of cancer in recent years is associated with a high rate of mortality. Numerous types of cancer have a low percentage of CD133(+) cells, which have similar features to stem cells. The CD133 molecule is involved in apoptosis and cell proliferation. The aim of this study was to determine the biological effect of CD133 suppression and its role in the chemosensitization of cancer cell lines. RT-PCR and immunocytochemical analyses indicated that CD133 was expressed in the cancer cell lines B16F10, MCF7 and INER51. Downregulation of CD133 by transfection with an antisense sequence (As-CD133) resulted in a decrease in cancer cell viability of up to 52, 47 and 22% in B16F10, MCF-7 and INER51 cancer cell lines, respectively. This decreased viability appeared to be due to the induction of apoptosis. In addition, treatment with As-CD133 in combination with cisplatin had a synergic effect in all of the cancer cell lines analyzed, and in particular, significantly decreased the viability of B16F10 cancer cells compared with each treatment separately (3.1% viability for the combined treatment compared with 48% for 0.4 µg As-CD133 and 25% for 5 ng/µl cisplatin; P<0.05). The results indicate that the downregulation of CD133 by antisense is a potential therapeutic target for cancer and has a synergistic effect when administered with minimal doses of the chemotherapeutic drug cisplatin, suggesting that this combination strategy may be applied in cancer treatment.

WT1 silencing by RNAi synergizes with chemotherapeutic agents and induces chemosensitization to doxorubicin and cisplatin in B16F10 murine melanoma cells.

The Wilm's tumor gene (WT1), encoding a transcription factor that modulates the expression of certain genes that are involved in proliferation and apoptosis, is overexpressed in numerous solid tumors. WT1 is important for cell proliferation and in the diagnosis of melanoma. The objectives of this study were to investigate whether WT1 silencing is capable of synergizing with chemotherapeutic agents and whether this silencing is capable of sensitizing cancer cells to doxorubicin and cisplatin in the B16F10 murine melanoma cell line. In the present study, B16F10 cells were simultaneously treated with median lethal doses (LD50s) of WT1-1 or WT1-2 small hairpin RNAs (shRNAs) and chemotherapeutic agents. A total of 24 h post-transfection, a [3-(4,5-dimethylthiazol-2yl)-2,5- diphenyl tetrazolium bromide assay] MTT assay was performed. To determine whether shRNA interference (shRNAi) is capable of sensitizing B16F10 cells to chemotherapeutic agents, cells were transfected with an LD50 of each of the recombinant plasmids, treated with varying concentrations of doxorubicin or cisplatin 24 h post-transfection, and analyzed 48 h later for inhibition of cell proliferation using the MTT assay. We observed that WT1-RNAi and the two chemotherapeutic agents acted synergistically to inhibit B16F10 cell proliferation. The greatest inhibition of cell proliferation was observed with the WT1-2/cisplatin (91%) and WT1-1/cisplatin combinations (85%). WT1 silencing using shRNAi induced the chemosensitization of cells to doxorubicin and cisplatin, with the greatest inhibition (85%) of cell proliferation being observed in the cells treated with the WT1-2/cisplatin 6 ng/µl combination. Our results provide direct evidence that WT1 gene silencing has a synergistic effect with chemotherapeutic drugs and sensitizes B16F10 melanoma cells to doxorubicin and cisplatin. This suggests that these combination strategies are potentially utilized in melanoma therapy.

Mouse mammary tumor virus-like gene sequences are present in lung patient specimens.

BACKGROUND: Previous studies have reported on the presence of Murine Mammary Tumor Virus (MMTV)-like gene sequences in human cancer tissue specimens. Here, we search for MMTV-like gene sequences in lung diseases including carcinomas specimens from a Mexican population. This study was based on our previous study reporting that the INER51 lung cancer cell line, from a pleural effusion of a Mexican patient, contains MMTV-like env gene sequences. RESULTS: The MMTV-like env gene sequences have been detected in three out of 18 specimens studied, by PCR using a specific set of MMTV-like primers. The three identified MMTV-like gene sequences, which were assigned as INER6, HZ101, and HZ14, were 99%, 98%, and 97% homologous, respectively, as compared to GenBank sequence accession number AY161347. The INER6 and HZ-101 samples were isolated from lung cancer specimens, and the HZ-14 was isolated from an acute inflammatory lung infiltrate sample. Two of the env sequences exhibited disruption of the reading frame due to mutations. CONCLUSION: In summary, we identified the presence of MMTV-like gene sequences in 2 out of 11 (18%) of the lung carcinomas and 1 out of 7 (14%) of acute inflamatory lung infiltrate specimens studied of a Mexican Population.